Bottom Line:
Although RsmB deficiency did not impact initiation from most codons, RsmD deficiency increased initiation from AUA, CAC and CAU (2- to 3.6-fold).Deletion of the S9 C-terminal tail resulted in poorer initiation from UUG, GUG and CUG, but in increased initiation from CAC, CAU and UAC codons (up to 4-fold).These observations suggest distinctive roles of 966/967 methylations and the S9 tail in initiation.

ABSTRACTThe accuracy of pairing of the anticodon of the initiator tRNA (tRNA(fMet)) and the initiation codon of an mRNA, in the ribosomal P-site, is crucial for determining the translational reading frame. However, a direct role of any ribosomal element(s) in scrutinizing this pairing is unknown. The P-site elements, m(2)G966 (methylated by RsmD), m(5)C967 (methylated by RsmB) and the C-terminal tail of the protein S9 lie in the vicinity of tRNA(fMet). We investigated the role of these elements in initiation from various codons, namely, AUG, GUG, UUG, CUG, AUA, AUU, AUC and ACG with tRNA(fMet(CAU) (tRNA(fMet) with CAU anticodon); CAC and CAU with tRNA(fMet(GUG); UAG with tRNA(fMet(CAU) ; UAC with tRNA(fMet(GUG) ; and AUC with tRNA(fMet(GUG) using in vivo and computational methods. Although RsmB deficiency did not impact initiation from most codons, RsmD deficiency increased initiation from AUA, CAC and CAU (2- to 3.6-fold). Deletion of the S9 C-terminal tail resulted in poorer initiation from UUG, GUG and CUG, but in increased initiation from CAC, CAU and UAC codons (up to 4-fold). Also, the S9 tail suppressed initiation with tRNA(fMet(CAU) lacking the 3GC base pairs in the anticodon stem. These observations suggest distinctive roles of 966/967 methylations and the S9 tail in initiation.

Mentions:
The pCATCACmetYGUG and pCATCAUmetYGUG constructs were introduced into E. coli SA and its derivatives to measure initiation from CAC:GUG and CAU:GUG codon:anticodon pairs, respectively. Transformants were grown in LB with Amp and streaked on LB-agar containing 100 μg ml−1 Amp (Amp100) as control and on LB-agar Amp100 with varying concentrations of Cm (Amp100 Cm100−250). Representative plates with Amp100 or Amp100 Cm250 (Figure 2A) show that the pCATCAUmetYGUG supported the growth of the strains deleted for methyltransferases in singles (ΔrsmB or ΔrsmD) or double (ΔrsmB ΔrsmD) (sectors 2–4) but not of the parent (sector 1), suggesting better initiation by tRNA in the absence of methylations at positions 966 and/or 967. In the plate assay, the transformants harbouring pCATCACmetYGUG (sectors 5–8) did not show a perceivable difference in growth (because of higher level of initiation from the matched CAC codon). However, as revealed by the CAT assays (Figure 2B, panels i and ii), deletion of methyltransferases in singles or double resulted in increased initiation by tRNA (∼2- to 3.6-fold) over the SA parent (C) from both CAC and CAU codons (see also Supplementary Figure S6). Interestingly, as shown in Figure 2B (panels iii and iv), the deficiency of these methyltransferases showed no significant changes in initiation with AUG (decoded by native tRNA) or UAG (decoded by near native tRNA).Figure 2.

Mentions:
The pCATCACmetYGUG and pCATCAUmetYGUG constructs were introduced into E. coli SA and its derivatives to measure initiation from CAC:GUG and CAU:GUG codon:anticodon pairs, respectively. Transformants were grown in LB with Amp and streaked on LB-agar containing 100 μg ml−1 Amp (Amp100) as control and on LB-agar Amp100 with varying concentrations of Cm (Amp100 Cm100−250). Representative plates with Amp100 or Amp100 Cm250 (Figure 2A) show that the pCATCAUmetYGUG supported the growth of the strains deleted for methyltransferases in singles (ΔrsmB or ΔrsmD) or double (ΔrsmB ΔrsmD) (sectors 2–4) but not of the parent (sector 1), suggesting better initiation by tRNA in the absence of methylations at positions 966 and/or 967. In the plate assay, the transformants harbouring pCATCACmetYGUG (sectors 5–8) did not show a perceivable difference in growth (because of higher level of initiation from the matched CAC codon). However, as revealed by the CAT assays (Figure 2B, panels i and ii), deletion of methyltransferases in singles or double resulted in increased initiation by tRNA (∼2- to 3.6-fold) over the SA parent (C) from both CAC and CAU codons (see also Supplementary Figure S6). Interestingly, as shown in Figure 2B (panels iii and iv), the deficiency of these methyltransferases showed no significant changes in initiation with AUG (decoded by native tRNA) or UAG (decoded by near native tRNA).Figure 2.

Bottom Line:
Although RsmB deficiency did not impact initiation from most codons, RsmD deficiency increased initiation from AUA, CAC and CAU (2- to 3.6-fold).Deletion of the S9 C-terminal tail resulted in poorer initiation from UUG, GUG and CUG, but in increased initiation from CAC, CAU and UAC codons (up to 4-fold).These observations suggest distinctive roles of 966/967 methylations and the S9 tail in initiation.

ABSTRACTThe accuracy of pairing of the anticodon of the initiator tRNA (tRNA(fMet)) and the initiation codon of an mRNA, in the ribosomal P-site, is crucial for determining the translational reading frame. However, a direct role of any ribosomal element(s) in scrutinizing this pairing is unknown. The P-site elements, m(2)G966 (methylated by RsmD), m(5)C967 (methylated by RsmB) and the C-terminal tail of the protein S9 lie in the vicinity of tRNA(fMet). We investigated the role of these elements in initiation from various codons, namely, AUG, GUG, UUG, CUG, AUA, AUU, AUC and ACG with tRNA(fMet(CAU) (tRNA(fMet) with CAU anticodon); CAC and CAU with tRNA(fMet(GUG); UAG with tRNA(fMet(CAU) ; UAC with tRNA(fMet(GUG) ; and AUC with tRNA(fMet(GUG) using in vivo and computational methods. Although RsmB deficiency did not impact initiation from most codons, RsmD deficiency increased initiation from AUA, CAC and CAU (2- to 3.6-fold). Deletion of the S9 C-terminal tail resulted in poorer initiation from UUG, GUG and CUG, but in increased initiation from CAC, CAU and UAC codons (up to 4-fold). Also, the S9 tail suppressed initiation with tRNA(fMet(CAU) lacking the 3GC base pairs in the anticodon stem. These observations suggest distinctive roles of 966/967 methylations and the S9 tail in initiation.